This grant is submitted as part of a package under the INTEGRATED PRECLINICAL/CLINICAL AIDS VACCINE DEVELOPMENT program [PAR-97-056]. The theme of the package is to determine the mechanism of protection in monkeys immunized with attenuated SHIV. It has been shown repeatedly that infection with attenuated SIV protects monkeys from challenge with virulent SIV. However the nature of the immune response(s) that are important for the protection remain undefined. Although live-attenuated HIV vaccines may never be safe enough for clinical use, understanding the mechanism by which attenuated viruses confer protection will provide critical information for use in designing safe and effective HIV Project will test the hypothesis that immunization of rhesus macaques with naked DNA constructs incorporating SHIV89.6 genes under the transcriptional control of novel promoters can protect animals from vaginal challenge with virulent SIV. The minimum viral components required for eliciting protection can be determined by immunizing monkeys with increasing complex mixtures of constructs which incorporate one or more SHIV89.6 viral genes, then challenging the monkeys with virulent SIV. Further, the use of non-lentiviral promoters to control expression may reduce the interval between immunization and protection from vaginal challenge with virulent SIV. Three specific aims are planned: Aim 1) Develop vectors and optimize nucleic acid vaccination methods for immunization with two or more antigen genes, Aim 2) Investigate the role that Immunostimulatory Sequences (ISS) in the DNA play in immune responses in primates and to determine if they can be used to enhance the immunity generated by the plasmid injections, Aim 3) To determine the minimal antigenic composition of a protective nucleic acid vaccine against SHIV89.6. Immune responses and protection induced by the env gene alone will be compared to those induced by a combination of env and one or more other viral genes. This project requests support for the preparation of the above listed vaccines and for initial testing in mice. The immune response analysis in primates and the challenge studies will be done using resources in Project 1.